Calcium ionophore, A23187, induced a dose dependent (IC50-5MuM) cardiotoxicity in cardiac myocytes in culture. A23187 also markedly potentiated the toxicity induced by daunomycin. This effect was seen both as a leakage of enzyme as well as in the reduction of ATP as well as leakage of [14]C-AMP in cells which have been labeled with [14]C-adenine. Electron-microscopic studies of these cells indicate the primary damage occurred in the mitochondria with little effect on the myofibrils. This early effect of ionophore and daunomycin was also seen on the beating rate which was markedly reduced in the cardiac myocytes. Verapamil, calcium channel blocker, potentiates the toxicity while another calcium channel blocker, nifedepine had no effect. Amrinone and milrinone which are bipyridine ionotropes markedly decreased the toxicity caused by daunomycin as determined by a decrease in the loss of ATP and enzyme leakage. Milrinone was found to be superior in this respect. Cyclic AMP has been implicated in the ionotropic effect as well as the cardiotoxicity induced by catecholamines. HL 725, a potent phosphodiesterase inhibitor, was found to have no protective effect on daunomycin-induced cardiotoxicity.